Micro titre plate

ABSTRACT

A micro titre plate comprising a number of wells filled with separation matrix. According to the invention the volume of the separation matrix is varied between at least some of the wells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 13/122,585, filed Apr. 5,2011 and was filed under 35 U.S.C. § 371 and claims priority tointernational patent application number PCT/SE2009/051113 filed Oct. 7,2009, published on Apr. 22, 2010 as WO 2010/044727, which claimspriority to application number 0802199-0 filed in Sweden on Oct. 15,2008.

FIELD OF THE INVENTION

The present invention relates to micro titre plates of the typementioned in the preamble of the independent claim 1 and to methods forhigh throughput screening of adsorption systems including methods fordetermination of adsorption isotherms.

BACKGROUND OF THE INVENTION

Multiwell plates, also called micro titre plates, have been used formany years in laboratories for the simultaneous analysis of a number ofsamples. Typical formats include 4, 24, 48, 96 and 384 wells per plate.Initially, these plates had solid bases and liquid samples were pipettedinto and out of the wells.

Subsequently, plates with wells provided with a lower well through hole(known as a “drip” if it is provided with downward protruding lips)pierced through the bottom surface. These micro titre plates allowed thesamples to flow through the wells which permitted larger sample volumesto be processed (since the sample size was no longer limited to thecapacity of the well).

Later developments of micro titre plates were provided with filter ormembrane wells in which each well was provided with a microporous filteror membrane which extended over the cross-section of the well such thatall of the sample passing through the well had to pass through thefilter or membrane. These micro titre plates are also called micro titrefilter plates.

A further development of a micro titre plate comprises wells with alower well through hole or drip and a filter or membrane and which wellsare each at least partly filled with a media such as a chromatographicgel or slurry or chromatographic particles.

Different screening processes that could be performed on such microtitre plates are for example screening of/for: i) conditions for optimalbinding capacity, ii) most efficient wash buffers for washing offimpurities from chromatography resin; iii) most efficient elutingsolution; iv) selectivity obtained using different ligands, v) bestresin either from capacity or purity perspective. Considering the multiwell format of microtiter plates, if processes under differentconditions need to be studied there are different possibilities tochange conditions on a single plate. For example the concentration ofthe sample added can be changed, the composition of the buffer in whichthe sample is dissolved can be changed, the effect of overall time ofcontact between the sample and a chromatography resin can be studied, orany combinations of the above.

High throughput studies of chromatographic separations using microtiterplates filled with chromatography resin proved highly efficient inreducing time and sample requirements necessary for development of largescale purification processes. While the studies reported have focused onspecific aspects of chromatographic steps, a very few studies have beenreported focusing on understanding physics of the separation processesstudied. From understanding the separation process perspective theknowledge of adsorption isotherm is of paramount importance. Adsorptionisotherms are fundamental property of any separation systems, and anisotherm should always be determined in order to full understandgoverning principles behind an adsorption process.

SUMMARY OF THE INVENTION

According to the present invention, an improved micro titre plate filledwith a separation matrix according to the characterising part of claim 1is provided.

Hereby the user can add the same volume of sample having the sameconcentration and still obtain data pertaining to different saturationof separation matrix with the sample because of the different mediavolumes in different wells.

Suitably the different media volumes are chosen based on a specificalgorithm assuring the highest possible quality of the data obtainedwith respect to describing of adsorption equilibria for the systemstudied.

Further suitable embodiments are described in the dependent claims.

Furthermore a method for determining an adsorption isotherm is providedaccording to claim 11.

Furthermore the use of a micro titre plate according to the inventionfor determining adsorption isotherms is described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic view of a micro titre plate filled with separationmatrix according to a first embodiment of the invention.

FIG. 2 is a schematic view of a micro titre plate filled with separationmatrix according to a second embodiment of the invention.

FIG. 3 is a schematic view of a micro titre plate filled with separationmatrix according to a third embodiment of the invention.

FIG. 4 shows an example of separation matrix volume distributions forthe plate layouts shown in FIG. 1-3.

FIG. 5a is a diagram showing the principle for obtaining an adsorptionisotherm using constant separation matrix volume.

FIG. 5b is a diagram showing the principle for obtaining an adsorptionisotherm using the present invention.

FIG. 6 shows the different separation matrix volumes of Example 1.

FIG. 7a shows the adsorption isotherm obtained using the six differentseparation matrix volumes of Example 1.

FIG. 7b shows the adsorption isotherm obtained using the eighthdifferent separation matrix volumes of Example 1.

FIG. 7c shows the adsorption isotherm obtained using the twelvedifferent separation matrix volumes of Example 1.

DETAILED DESCRIPTION OF THE INVENTION

A multiwell plate, also called a micro titre filter plate or micro titreplate, that is filled with a separation matrix is provided. Theseparation matrix could be a chromatography media and it can also becalled for example a solid phase, resin, gel or adsorber. The number ofwells in the plate is in a first embodiment of the invention 96. Othernumbers are however also possible such as 24, 48, and 384. According tothe invention at least some of the wells of the plate are filled withwell defined different amounts of separation matrix. The distribution ofseparation matrix in the different wells can be different for differentuses of the plate. Micro titre plates are used for the screening ofdifferent chromatographic attributes such as for example determinationof binding capacities, effectiveness of wash buffer in removal ofimpurities, effect of different buffer conditions on selectivity (arelative difference between capacities for different solutes adsorbingon the solid phase) or for studying the effect of solute concentrationin the liquid phase on the amount of solute adsorbed on the solid phase,a so called adsorption isotherms studies. For a person skilled in theart of chromatography it is known that adsorption isotherm is afundamental concept for describing adsorption processes, including allthe processes listed above. According to the invention it has been shownthat for the study of adsorption isotherm it is beneficial to have adistribution of different volumes of separation matrix in at least someof the different wells, A correctly chosen distribution of differentvolumes in a plate enables collection of adsorption data which qualitywill be sufficient to describe an adsorption isotherm in the mosteffective manner using the smallest number of data points necessary.

A first embodiment of the invention is shown in FIG. 1. In this examplea 96 wells plate is used and there are six different volumes ofseparation matrix in the wells. The two first columns have in thisexample the same volume of separation matrix in all wells. The two nextcolumns have the same volume of separation matrix in each well but thisvolume is different from the first volume etc. according to FIG. 1.

FIG. 2 is another example of an advantageous distribution. Here thereare 12 different volumes of separation matrix. The volume of separationmatrix is the same in all wells in the same column. But the volumediffers between the columns according to FIG. 2.

FIG. 3 is a further example of distribution. Here each row has the samevolume of separation matrix in all its wells and all rows have differentvolumes of separation matrix.

FIG. 4 is an example of separation matrix volume distributions for theplate layouts shown in FIG. 1-3. These numbers are only meant to be usedas an example and should not be limiting the scope of the invention.

Adsorption isotherm describes thermodynamic of adsorption processstudies, and therefore, is a fundamental property of any proteinseparation system. This knowledge about the type of isotherm responsiblefor a separation is a prerequisite to correctly describe the separation.

Among many methods for determination of adsorption isotherms a methodbased on batch adsorption is fairly popular. In this method a welldefined volume of separation matrix and sample are brought in contactwith each other and after equilibrium is reached between concentrationsin the liquid and solid phases these concentrations are measured and asingle point on the adsorption isotherm is obtained. In order to obtainmore points either ratio of separation matrix to sample volume or sampleconcentration need to be changed. While changing sample volume is fairlyeasy there may be limitations related to a total volume of a systemavailable for experiments. Changing separation matrix volume is alsodifficult and special methods need to be developed to have a fullcontrol over the resin volume used. Changing sample concentration seemseasy but only if composition of bulk liquid is known. If the compositionis complex, changing sample concentration without changing thecomposition is close to impossible for most of practical situations,especially if the concentration needs to be increased. FIG. 5a shows adiagram of an adsorption isotherm achieved by using the same solid phasevolume and changing the sample concentration. The x-axis isconcentration in liquid phase and the y-axis is concentration in solidphase. From each starting point with different sample concentration asingle point on the adsorption isotherm is achieved when equilibrationis reached. The operating lines shown in FIG. 5a are a graphicalrepresentation of mass balance over a single well. The operating lineoriginates at the point representing initial state of the system andends at the point representing the equilibrium state given by theadsorption isotherm. The phase ratio, Vliq/Vsolid is the slope of theoperating line.

According to the invention plates are provided with different separationmatrix volumes in the wells as described above. In FIG. 5b the method ofachieving an adsorption isotherm from such a plate is shown. The samesample concentration and volume is used all the time but differentpoints on the adsorption isotherm will still be achieved because of thedifferent operating lines originating from the fact that the solid phasevolume is different.

From mathematical perspective it can be shown that adsorption isothermsare best described by physical models if points on the adsorptionisotherm are distributed in a certain fashion. One example could be thateach consecutive equilibrium point is obtained at 2 times higherconcentration than the preceding point, and that at least 10% of allpoints should describe isotherm in the region where capacities are lowerthan 50% of maximum capacity.

Another example of how to distribute the volumes of separation matrix inthe micro titre plate can be to provide volumes that when the plate isused for adsorption isotherm measurements with constant volume andconcentration of added sample to the wells that will be used will resultin an isotherm where concentrations in the liquid phase after the end ofthe experiment will be distributed according to the followingrelationship: concentration in liquid phase in one well divided with theconcentration in liquid phase in a neighbouring well with differentamount of separation matrix will be between 1 and 3 and most preferable2. When the larger volume is the numerator. See further example 1 below.

According to the invention a method for determining an adsorptionisotherm is further provided. The method comprises the steps of:

-   -   using a micro titre plate according to the invention as        described above;    -   adding the same volume and concentration of a sample to the        number of wells needed for the analysis;    -   waiting for a predefined period of time, preferably until        equilibrium is reached;    -   measuring concentration of liquid phase and/or solid phase in at        least two wells with different volumes of separation matrix.

Furthermore, this invention covers the use of a micro titre plateaccording to the invention for determining adsorption isotherms.

Furthermore a method of filling a micro titre plate with separationmatrix is proposed in this invention. The method comprises the steps of:

-   -   determining a suitable distribution of volumes of separation        matrix between the wells based on the specific screening process        that will be performed;    -   filling the wells of the micro titre plate with separation        matrix according to the determined suitable distribution.

These plates according to the invention would also be advantageous touse in other screening processes. For example in determination of effectof contact time on binding capacities. Such studies will lead toelucidation of transport mechanism behind protein adsorption, which inturn would lead to ability to mechanistically describe the adsorptionprocess studied.

EXAMPLES

The present examples are provided for illustrative purposes only, andshould not be construed as limiting the scope of the present inventionas defined by the appended claims. All references given below andelsewhere in the present specification are hereby included herein byreference.

Example 1

From a statistical point of view it is recommended that in order toobtain the most precise values of model parameters describing anadsorption isotherm, batch experiments should be performed applying anexperimental design that would result in equilibrium concentrationscharacterized by a dilution like (geometric) pattern. This can be shownusing the following as an example a case of a Langmuir isotherm, givenhere by Eq.(1), with maximum binding capacity, Qmax, and thedissociation constant, K, as model parameters. Following the work ofCurrie (1982) who performed extensive testing of the typical designsused to evaluate enzyme kinetic parameters for the Michaelis-Menten typeof enzyme kinetics and showed that the modified dilution/geometricdesign resulted in the most precise values of the model parameters, itcan be concluded that similar designs would be applicable for theLangmuir isotherm model because from the mathematical point of view, theequation describing Michaelis-Menten kinetics is not different from theLangmuir isotherm.

$\begin{matrix}{{q(C)} = \frac{Q_{\max}C}{K + C}} & (1)\end{matrix}$

According to Currie (1982), for a sequence of n observations obtainedfrom runs performed at locations described by the form given by Eq.(2),the values of parameters in Eq.(1) that minimise the generalisedparameter variance are shown in Table 1.C_(i)=ar^(i−1)  (2)where: C_(i) stands for the concentration of protein in the i-th run,and the a and r are the parameters.

TABLE 1 n a r 6 0.601 K 3.45 8 0.508 K 2.52 12 0.434 K 1.84

In case of a typical adsorption isotherm experiments using a batchadsorption protocol the following mass balance always holds at each runlocationq _(i)(C _(i))=β(C _(0,i)−C _(i))  (3)where: βis a phase ratio defined as V_(L)/V_(S); V_(L) and V_(S)represents volume of liquid and solid phase, respectively; q_(i)(C_(i))is an equilibrium adsorption capacity for i-th run, C_(o,i) and C_(i)are initial and equilibrium protein concentrations for i-th run.

Combining Eq.(1) and Eq.(2) and assuming that the sample volume isconstant yields Eq.(4) which describes the preferred distribution ofphase ratios for a constant sample concentration, C₀ case, as a functionof number of run locations and the expected shape the adsorptionisotherm.

$\begin{matrix}{V_{{solid},i} = {V_{samp}\left\lbrack {\frac{1}{C_{0} - {ar}^{i - 1}}\frac{Q_{\max}{ar}^{i - 1}}{K + {ar}^{i - 1}}} \right\rbrack}^{- 1}} & (4)\end{matrix}$

In order to obtain a suitable distribution of V_(solid), an a prioriknowledge of values for the dissociation constant K and the maximumequilibrium capacity Q_(max) is needed. Obviously, this is an evidentparadox since these parameters are to be found using the above design.In order to circumvent this situation, educated guesses for theseparameters need to be used. These guesses, can be based on datapertaining to similar adsorptive systems.

Examples of suitable resin distributions for the case when system volumeis 200 uL and initial concentration is 4 g/L for a fairly typicalprotein adsorption characterize by Q max=50 g/L, and K=0.01 g/L is givenin Table E1 of FIG. 6. The isotherm that would be obtained using resindistributions form Table E1 are shown in FIGS. 7a, b and c.

The above experimental design can be further simplified, in thesimplified version the design is based on geometrical series with afactor of 2, and with the second run location being performed at aninitial concentration yielding the equilibrium concentration to be equalto the value of dissociation constant K. This design is thuscharacterize by the following relations (Eq.(5)), which uponsubstitution into Eqn.(1) and (2)characterize, will yield the newdistribution of the solid phase.C_(i)=Ka^(i−2)  (5)

Following a similar logic the following modification to the simplifieddesign can be also proposed, especially if the value of K is not reallyknown.

$\begin{matrix}{C_{i} = \frac{C_{\max}}{2^{i - 1}}} & (6)\end{matrix}$where: C_(max) is the highest available concentration of protein, and iis the run number.

Even though the present invention has been described above in terms ofspecific embodiments, many modification and variations of this inventioncan be made as will be obvious to those skilled in the art, withoutdeparting from its spirit and scope as set forth in the followingclaims.

What is claimed is:
 1. A micro titre plate comprising a number of wellsdisposed in a plurality of columns and rows, the wells being filled withthe same type of separation matrix, wherein the separation matrix in thewells of the plate has a series of volumes V₁, V₂, . . . V_(n)distributed according to a specific pattern that does not obeylogarithmic or a single degree freedom linear relationship and sameseparation matrix volume is provided in all the wells of either the samecolumn or the same row in the plate, and wherein when same volume andconcentration of a liquid sample is added to each of the wells, atequilibrium, the concentration of liquid phase in one well divided bythe concentration of liquid phase in a neighbouring well with differentamount of separation matrix is between 1 and
 3. 2. A method of fillingmicro titre plate of claim 1 with separation matrix, the methodcomprising, determining the specific distribution pattern of volumes ofseparation matrix between the wells based on the specific screeningprocess that will be performed; and filling the wells of the micro titreplate with separation matrix according to the determined specificdistribution pattern.
 3. A method for determining an adsorption isothermcomprising the steps of: using the micro titre plate of claim 1; addingthe same volume and concentration of a liquid sample to each of thenumber of wells needed for the analysis; waiting for a predefined periodof time until at least some of the sample is adsorbed onto theseparation matrix; and measuring concentration of the sample remainingin the liquid phase and/or solid phase in at least two wells withdifferent volumes of separation matrix.
 4. The micro titre plate ofclaim 1, wherein the series of volumes V₁, V₂, . . . V_(n) isdistributed according to a specific pattern for the calculation of anadsorption isotherm of the separation matrix such that the series ofvolumes results in a series of corresponding isotherm data points in thecalculation of the adsorption isotherm and the volumes of the separationmatrix is distributed such that at least 10% of the isotherm data pointsobtained being below the 50% maximum capacity levels of the adsorptionisotherm.
 5. The micro titre plate of claim 4, wherein each consecutiveisotherm data point is obtained at 2 times higher concentration than thepreceding point.
 6. The micro titre plate of claim 1, wherein each wellin the plate has a permeable filter in the bottom.
 7. The micro titreplate of claim 1, wherein the series of volumes V₁, V₂, . . . V_(n) ofthe separation matrix is distributed according to FIG. 1 and FIG.
 4. 8.The micro titre plate of claim 1, wherein the series of volumes V₁, V₂,. . . V_(n) of the separation matrix is distributed according to FIG. 2and FIG.
 4. 9. The micro titre plate of claim 1, wherein the series ofvolumes V₁, V₂, . . . V_(n) of the separation matrix is distributedaccording to FIG. 3 and FIG.
 4. 10. The method of claim 3, wherein theconcentration of liquid phase in one well divided with the concentrationof liquid phase in a neighbouring well with different amount ofseparation matrix is between 1 and
 3. 11. The method of claim 3, whereinthe concentration of liquid phase in one well divided with theconcentration of liquid phase in a neighbouring well with differentamount of separation matrix is about
 2. 12. The method of claim 3,wherein the predetermined waiting time is until an equilibrium betweenthe liquid and solid phases has been reached.